The long term objective of this proposal is to understand the factors that contribute to the virulence and pathogenicity of Bacteroides fragilis, the most important anaerobic pathogen in suppurative intra-abdominal and pelvic infections in human beings. Since this requires a genetic and physiological analysis of the virulence factors, our approach has been to develop new genetic systems for use in B. fragilis in order to identify potential virulence determinants. We are focusing on the aerotolerance of B. fragilis as an important factor in the organisms' ability to survive in oxygenated tissue. We will analyze a set of B. fragilis proteins that are highly expressed only in the presence of oxygen. We will study the control of the synthesis of these oxygen response proteins (ORPs) and attempt to identify global and gene specific regulators. We will also study the process of the shut-off of most B. fragilis proteins normally made during anaerobiosis that occurs when the growing cells are challenged with oxygen. We will continue our studies on the superoxide dismutase of B. fragilis and determine its role in B.fragilis survival in vitro and in vivo. Since we have determined an important role for the B.fragilis neuraminidase in the ability of B.fragilis to grow in two in vivo test systems, we will concentrate on defining the control region of the nanH structural gene and in understanding how its expression is regulated. We will use an adaptation of the TnphoA system that has been used in E.coli to identify exported proteins, in order to study surface proteins of B. fragilis. Since many cell envelope proteins play a role in bacterial pathogenicity, we will use TnCef to identify virulence determinants among B. fragilis surface or exported proteins. Mutants with alterations in potential virulence factors will be tested for their ability to grow in two model systems of B. fragilis infection, the rat granuloma pouch, and CHO or Monika P15 animal cells in culture. We will search for B. fragilis genes that are only expressed when the bacteria are growing in vivo. These genes are likely to play an important role in the establishment and maintenance of B. fragilis infections. To further define the initial steps in B.fragilis infection of the rat pouch, we will perform a histological analysis of the infected pouch at early times after infection. We will determine the location of the infecting B. fragilis cells during the establishment of the anaerobic environment that is required for bacterial growth.